This invention relates to the field of nucleic acid detection and, more specifically, to the processing of samples to release nucleic acids in a condition suitable for direct detection.
Nucleic acid detection through modern molecular biological techniques has revolutionized diagnosis of infections, cancer, inborn genetic errors, HLA typing, and forensic and paternity testing. Methods to detect nucleic acids commonly requires several sample processing steps, including use of a lysis reagent to lyse cells and release the nucleic acids contained within the cells. Lysis reagents typically consist of a strong detergent such as sodium dodecyl sulfate and alkaline pH conditions.
The need for multiple processing steps when using a lysis reagent, such as one containing a strong detergent, primarily results from inhibitors of later nucleic acid detection steps that are present or associated with the lysis reagent. The inhibitors must be neutralized or removed before amplification or other additional steps in nucleic acid detection can proceed. These additional steps result in increased labor and materials costs for the clinical laboratory. Use of a lysis reagent for nucleic acid detection also is detrimental because it can, under some circumstances, degrade the nucleic acids, thereby decreasing sensitivity in some assay formats. Thus, a need exists for an approach to isolate nucleic acids from a cell sample that avoids the additional steps associated with lysis reagents and allows for release and detection from a single reagent addition step.
Accordingly, it is an object of the present invention to eliminate the additional processing steps and degradation associated with nucleic acid lysis procedures. This is achieved by using lipids that are non-denaturing for enzymes and proteins required in further processing steps.
It is also an object of the present invention to provide compositions for releasing nucleic acid from cells or samples that include reagents for labeling or performing amplification such that release and detection of nucleic acid can be performed by a single reagent addition step.
To accomplish these and other objectives, there has been provided, according to one aspect of the present invention, a composition comprising an aqueous solution for releasing nucleic acid from a sample for direct detection, comprising one or more lipids and, one or more of: i) an enzyme(s) to degrade cell structure; ii) a non-ionic membrane fluidizing compound(s); and iii) a metal chelator(s). The aqueous solution is non-inhibitory of enzymes or proteins that are used in nucleic acid release, amplification, labeling or detection, and can include one or more nucleic acid probes or primers complementary to the nucleic acid to be detected.
According to one embodiment of the present invention, the lipids of the aqueous solution comprise lipids in the form of liposomal vesicles or other structure for encapsulating the aqueous solution.
According to another embodiment of the present invention, the aqueous solution includes reagents for labeling nucleic acid. Such reagents comprise a compound comprising a photoactivatible binding ligand, a label comprising a detectable moiety and, optionally, a nucleic acid binding enhancer moiety.
According to yet another embodiment of the present invention, the aqueous solution further comprises one or more nucleic acid probes or primers complementary to the nucleic acid to be detected.
According to still yet another embodiment of the present invention, the one or more lipids of the aqueous solution comprise 3-(2-aminopropyl-1,3-dihexadecyloxypropyl) hexadecyl ether, 3-(2aminopropyl-1-octadecyloxy-3-benzyloxypropyl) benzyl sulfide, or bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-propyl amine)-polyethyleneglycol.
In another aspect of the present invention, there is provided a composition comprising an aqueous solution comprising one or more membrane fluidizing compounds for releasing nucleic acid and one or more of: i) an enzyme(s) to degrade cell structure; ii) a lipid(s); and iii) a metal chelator(s). The aqueous solution is non-denaturing and non-inhibitory of enzymes or proteins that are used in nucleic acid release, amplification, labeling or detection.
According to one embodiment of the present invention, the lipids of the aqueous solution comprise lipids in the form of liposomal vesicles or other structure for encapsulating the aqueous solution.
According to another embodiment of the present invention, the aqueous solution includes reagents for labeling nucleic acid. Such reagents comprise a compound comprising a photoactivatible binding ligand, a label comprising a detectable moiety and, optionally, a nucleic acid binding enhancer moiety.
According to yet another embodiment of the present invention, the aqueous solution further comprises one or more nucleic acid probes or primers complementary to the nucleic acid to be detected.
In accordance with another another aspect of the present invention, methods are provided for detecting the presence of a nucleotide sequence in nucleic acid of a sample using the aqueous solutions comprising a lipid or membrane fluidizing compound containing compositions of the present invention. Such methods are applicable to clinical specimens and are useful for diagnosing a variety of diseases and conditions.
In accordance with still yet another aspect of the present invention, kits are provided for releasing nucleic acid from a sample in a form suitable for directly detecting the nucleic acid. The kit comprises a vial containing an aqueous solution comprising one or more lipids for releasing nucleic acid from the cells and further comprising one or more of an enzyme(s) to degrade cell structure, a non-ionic membrane fluidizing compound(s) and a metal chelator(s). The aqueous solution is non-denaturing and non-inhibitory of enzymes or proteins used in nucleic acid release, amplification, labeling or detection.
In one embodiment, the kit further comprises or more nucleic acid probes or primers complementary to the nucleic acid to be detected, wherein said probes or primers are contained in the vial with the aqueous solution or are contained in one or more separate vials.
In another embodiment, the kit includes a means to prepare liposomes with the reagents supplied with the kit. In another embodiment, the kit further includes reagents for labeling nucleic acid, wherein said reagents are contained in the vial with the aqueous solution or are contained in one or more separate vials.
The present invention provides novel compositions and methods for processing cell samples under conditions such that nucleic acids in cells or otherwise inaccessible to detection are released in a form suitable for direct nucleic acid detection assays. The compositions of the invention are designed to release nucleic acid from cells under conditions that do not result in denaturation of enzymes or proteins used in nucleic acid release, amplification, labeling or detection.
General Definitions:
Oligonucleotide: Low molecular weight deoxyribo-, ribo-, copolymers of deoxyribo- and ribonucleic acids of chain lengths between 3 and 150. Such oligonucleotides can have modified nucleotide residues such asxe2x80x94Oxe2x80x94methoxy, phosphorothio-, methylphosphonates and others known in art.
Primers: Usually oligonucleotides which are used for extension reaction by a nucleic acid polymerase after a template primer hybrid is formed. Such primers can carry sequences specific for transcription by an RNA polymerase.
Nucleic Acid Probe: Nucleic acid with substantially complementary sequences to the target nucleic acids for detection or capture from a mixture. Such probes can be labeled for detection or immobilized onto a solid support to enrich the target by capture. A probe can be an single stranded or partially double stranded and can be an oligonucleotide or a larger nucleic acid.
Membrane fluidizing compound: A chemical substance that renders a cell membrane fluid or flexible to facilitate release of cellular material into solution or uptake of extracellular contents. Compounds that induce pinocytosis in addition to fluidizing the membrane also are included within the meaning of a membrane fluidizing compound as used herein. A membrane fluidizing compound can be a lipid or a non-lipid and can be ionic or non-ionic. Membrane fluidizing compounds generally do not cause cell death at lower concentrations that effect membrane fluidity, however, cell death typically results at higher concentrations of the compound.
Lipid: Any of various substances that are soluble in non-polar organic solvents (such as chloroform and ether), that with proteins and carbohydrates constitute the principal structural components of living cells, and that include fats, waxes, phosphatides, cerebrosides, and related and derived compounds.
Liposome vesicles: A vesicle composed of one or more concentric phospholipid bilayers. The structure of the liposomes may be as a multilamellar vesicle (MLV), a small unilamellar vesicle (SUV), a large unilamellar vesicle (LUV). A liposome is formed from a single lipid or combination of lipids (i.e., lipsosmal formulation) and optionally other compounds.
Thiocationic lipid: A lipid molecule with sulfur substitution and which is positively charged at neutral pH.
Photoreagent or photoactive reagents: Reagents which under appropriate wavelengths of light exposure form a covalent bond with nucleic acid.
Preferred Embodiments:
A composition of the present invention for releasing nucleic acid from a cell sample in a form suitable for directly detecting the nucleic acid comprises an aqueous solution comprising one or more lipids for releasing nucleic acid from the cells. As used herein, an aqueous solution is a water and/or other water miscible solvent and further includes a buffer to stabilize the pH between 4 and 11, with the ultimate pH depending on the stability of the nucleic acid to be released.
The aqueous solution comprising one or more lipids includes those lipids suitable for releasing cellular or otherwise inaccessible nucleic acid without denaturation. Liposomal formulations containing cationic lipids that have been used for delivery of oligonucleotides and other agents to target cells are useful for releasing nucleic acid from cells without denaturation as provided herein. PCT WO 96/40627 and U.S. Pat. Nos. 5,851,548, 5,759,519, 5,756,352, and 5,739,271 teach liposomal formulations containing cationic lipids.
The lipids used in the present compositions for releasing nucleic acid from cells include complex mixtures of different lipophilic substituents. Such complex mixtures allow for optimization of the physical properties of the liposomes, such as pH sensitivity, temperature sensitivity and size. For example, in certain embodiments, dioleoylphosphatidylethanolamine (xe2x80x9cDOPExe2x80x9d), and other pH sensitive amphiphilic compounds can be used to formulate liposomes which destabilize at acidic pH. This promotes fusion of the liposome with endosomal membranes when exposed to the degradative acidic pH and enzymatic contents of the endosome, resulting in release of the contents of the endosome into the cytoplasm. (Ropert, et al., Biochem. Biophys. Res. Comm. 183(2):879-895 (1992); Juliano, et al., Antisense Res. and Dev. 2:165-176 (1992)). Although not wishing to be bound by any particular theory, it is believed that pH controlled degradation of liposomes in the cytoplasm of the cell enhances release of nucleic acids.
Lipids used in the present compositions for releasing nucleic acid from cells also can include sterols to enhance stability of liposomal vesicles both in vitro and in vivo. In particular, organic acid derivatives of sterols, such as cholesterol or vitamin D3, which have been reported to be easier to formulate than their non-derivatized water-insoluble equivalents (U.S. Pat. Nos. 4,721,612 and 4,891,208), are useful in preparing liposomal formulations as described herein.
Preferred lipids for use in the present compositions and methods are cationic lipids (i.e., derivatives of glycerolipids with a positively charged ammonium or sulfonium ion-containing headgroup), including those useful in liposomal formulations for the intracellular delivery of negatively charged biomolecules such as oligonucleotides. The usefulness of cationic lipids may be derived from the ability of their positively charged headgroups to interact with negatively charged cell surfaces, although this is not known for certain. The cationic lipid N-(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (xe2x80x9cDOTMAxe2x80x9d) as described by Felgner, et al., Proc. Natl. Acad. Sci. (U.S.A) 84:7413-7417 (1987) (U.S. Pat. No. 4,897,355) is a cationic lipid with an ammonium group that can be used in liposomal formulations present in the compositions of the invention. In such formulations, DOTMA may bind to DNA through an ionic lipid-DNA complex that assists in releasing nucleic acid from a cell. Other ammonium ion-containing cationic lipid formulations that can be used in the nucleic acid releasing compositions of the present invention include the DOTMA analog, 1,2-bis(oleoyloxy)-3(trimethylammonio)propane (xe2x80x9cDOTAPxe2x80x9d) (Stamatatos, et al., Biochem., 27:3917-3925 (1988)); the lipophilic derivative of spermine (Behr, et al., Proc. Natl. Acad. Sci. (U.S.A), 86:6982-6986 (1989)); and cetyltrimethylammonium bromide (Pinnaduwage, et al., Biochem. Biophys. Acta, 985:33-37 (1989); Leventis, et al., Biochem. Biophys. Acta, 1023:124-132 (1990); Zhou, et al., Biochem. Biophys. Acta, 1065:8-14 (1991); Farhood, et al., Biochem. Biophys. Acta, 1111:239-246 (1992); and Gao, et al., Biochem. Biophys. Res. Comm., 179:280-285 (1991)).
Cationic lipids are commercially available including DOTMA (Gibco BRL, Bethesda, Md.), DOTAP (Boehringer Mannheim, Germany), and 1,2-diacyl-3-trimethylammonium propane (xe2x80x9cTAPxe2x80x9d) (Avanti Polar Lipids, Alabaster, AL).
Cationic lipids containing sulfonium ions (i.e., thiocationic lipids) also can be used in the present nucleic acid releasing compositions. Sulfonium ions have entirely different physical properties than ammonium ions, which provides sulfonium cationic lipids with some unique properties. Ammonium ion-containing compounds are classified as hard bases, because the nitrogen atom possesses high electronegativity, is difficult to polarize and oxidize, and the valence electrons are held tightly by the nucleus. This characteristic may account for some of the toxicity associated with ammonium ion-containing lipid formulations. In contrast, sulfonium ion-containing compounds are classified as soft bases, because the sulfur atom possesses low electronegativity, is easy to polarize and oxidize, and the valence electrons are held more loosely by the nucleus. This decreased charge density exhibited by sulfonium ion-containing (i.e. xe2x80x9cthiocationicxe2x80x9d) lipids may effectuate an enhanced interaction with negatively charged cellular membranes, as well as a decreased toxicity, leading to compositions with increased ability to release cell nucleic acid in a non-denatured form.
Cationic lipids with relatively small polar headgroups as described by Feigner, et al., J. Biol. Chem., 269(4):2550-2561 (1994), can be particularly useful in the present compositions for releasing nucleic acids. However, the sulfonium ion type cationic lipid, which has a relatively larger headgroup, also can be useful because of the physiochemical properties associated with the sulfonium ion. A lipid headgroup that consists of a sulfur atom surrounded by adjoining saturated carbon atoms exhibits a diffusion of charge to the neighboring carbon atoms that can facilitate interaction of the lipid with cellular membranes, as well as decrease the toxicity of the lipid (U.S. Pat. No. 5,759,519).
Liposomal preparations of the present invention can have a positively charged surface by including in the formulation, saturated or unsaturated aliphatic amines, including, for example, stearylamine and oleylamine, sphingosine, phosphatidylethanolamine, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride, cholesterylhemisuccinate, 3B-(N-(Nxe2x80x2,Nxe2x80x2-dimethylaminoethane)carbamoyl)cholesterol and cholesterol(4xe2x80x2-trimethylammonio)butanoate, with preference given to stearylamine and sphingosine as described in U.S. Pat. No. 5,759,519.
The present compositions for releasing nucleic acid include a lipid that can form liposomes or other structures under the appropriate conditions. Prior methods of forming liposomes and encapsulating aqueous solution are applicable for preparing the nucleic acid releasing compositions of the present invention (e.g., Olson, et al., Biophys, acta, 557:9 (1979)). For example, prior art liposomal formulations used to encapsulate hemoglobin (e.g., U.S. Pat. No. 4,911,929) are to produce liposomal vesicles as described herein. Such liposomal formulation contains roughly equivalent quantities of cholesterol and phosphatidylcholine, with 5 to 10% negatively charged lipid, such as phosphatidic acid, dicetyl phosphate, or dimyristoyl phosphatidyl glycerol (DMPG). Hydration of the dried lipid film results in formation of multilamellar vesicles (MLV), which can be extruded at low-pressure (e.g., 50-90 psi) through filters of progressively smaller pore size to large unilamellar vesicles (LUVs). Once the liposomal vesicles are formed, any unencapsulated aqueous solution can be removed, if desired, by centrifugation or diafiltration and then recycled.
Lipid used for the formation of the liposome can be natural or synthetic and include phospholipids, glycolipids, and lipid related compounds. Exemplary lipids include, lecithin (phosphatidylcholine), phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiolipin, and hydrogenated derivatives thereof, which can be used either alone or in combination. The glycolipids include cerebroside, sulfolipid (e.g., sulfatide), and ganglioside. The structure of the liposomes may be as a multilamellar vesicle (MLV), a small unilamellar vesicle (SUV), or large unilamellar vesicle (LUV).
To stabilize the lipid, an antioxidant such as tocopherol (vitamin E) can be added to the solution. A suitable amount of an antioxidant is about 0.01 to 0.5% by weight based on the weight of the phospholipid. The liposome composition of the invention also can contain as a stabilizer, a high molecular weight polymer such as albumin, dextran, vinyl polymers, non-ionic surface active agents, gelatin, and hydroxyethyl starch.
Liposomal vesicles that encapsulate aqueous solutions as used herein can be prepared by a variety of known methods. For example, conventionally used hydration, reversed phase evaporation, removal of surfactant, solvent injection, freeze-thawing and dehydration-rehydration can be employed.
In the hydration method, the selected lipids are dissolved in an organic solvent (e.g., chloroform and ether), which is non-denaturing, and the solvent is evaporated from the resulting solution yield a thin homogeneous film. The aqueous solution containing, for example, an enzyme(s), a non-ionic membrane fluidizing compound(s), a metal chelator(s) or nucleic acid probes or primers (discussed further below) is added to the thin membrane, and the mixture is subjected to agitation and sonication to yield a liposome preparation encapsulating the aqueous solution. The aqueous solution contains a buffer at a pH between 4 and 11. The pH of the buffer is chosen such that when the lipids or liposomes are added to an assay medium, the final pH in a range suitable to preserve nucleic acids in solution.
In the reversed-phase evaporation method, the selected lipids are dissolved in an organic solvent (e.g., chloroform and ether), as discussed above, and are added to the aqueous solution and subjected to agitation, sonication and high pressure homogenization to uniformly disperse the aqueous solution. The solvent is evaporated from this dispersion to yield a liposome preparation encapsulating the aqueous solution.
In the removal of surfactant approach, the selected lipids dissolved in organic solvent are mixed with a surfactant (e.g., cationic surfactant such as cholic acid or deoxycholic acid, and a non-ionic surfactant such as Triton X-100 and octyl-D-glucoside) and added to the aqueous solution, which is followed by agitation, sonication and high pressure homogenization to uniformly disperse the aqueous solution. The surfactant is then removed by dialysis, gel filtration and ultrafiltration, which are applied singly or in combination.
In the solvent injection, approach, the selected lipids are dissolved in organic solvent and are added to the aqueous solution, which has been set for a temperature about 10xc2x0 C. higher than the boiling point of the organic solvent. Then, the organic solvent is evaporated.
The aqueous solution of the present nucleic acid releasing compositions also can include, for example, substances other than lipids that enhance release of nucleic acid depending on the nature of the sample and the environment in which the nucleic acid is contained (e.g., the type of cell). Such nucleic acid releasing substances include, for example, an enzyme(s) to degrade cell structure, a non-ionic membrane fluidizing compound(s), and/or a metal chelator(s).
Enzymes suitable for use with lipid containing aqueous solution are available from natural sources or produced by recombinant DNA methods. Such enzymes include, for example, lysozyme, lipases, and proteinases such as proteinase K, pronase, trypsin and chymotrypsin. Lysozymes from bovine, chicken, human and lipases from wheat germ, human, yeast and other sources also are suitable enzymes to degrade cell structure. These enzymes preferably are nuclease free to support stability of released nucleic acids in solution. The aqueous solution containing lipids and enzymes for releasing nucleic acid can be encapsulated into a liposome, if desired.
The enzymes are used at a molar ratio of lipid to enzyme of between 10,000:1 and 1:10,000. The optimal ratio of enzyme to lipid can be readily determined by one skilled in the art. This can be accomplished by mixing target cells with various lipid:enzyme ratios, and determining the effectiveness of releasing nucleic acid in a probe hybridization assay.
Non-ionic membrane fluidizing compounds, which have been described in Suciu et al., Mol. Microbiol., 21:181-95 (1996), Nabekura et al., Pharm Res., 13(7):1069-72 (1996), and Lindow et al., Cryobiol., 32(3):247-258 (1995), and include aromatic alcohols such as all phenyl, napthyl, and higher alcohols, also can be used to release nucleic acid from cells without denaturation of enzymes or proteins. The hydrocarbon side chains of aromatic alcohols can be from C1 to C50 and longer, preferably between C1 and C20. Thexe2x80x94OH residue can be at the Cn terminus carbon for a primary alcohol or any place as in a secondary or tertiary alcohol. The Cxe2x80x94C bonds in Cn chain in addition to single bond can have unsaturated linkages in the form of double or triple bonds. The carbon chain also can have secondary and tertiary C-linkages. Phenethyl alcohol, sec-phenethyl alcohol, benzyl alcohol are examples of non-ionic membrane fluidizing compounds.
Non-ionic membrane fluidizing compounds can be included in the aqueous solutions of the present invention provided they enhance release of nucleic acids from cells without creating an enzyme or protein inhibitory environment. Such compounds can be present in the aqueous solution at a concentration between 0.001% and 10.0%. The final concentration of non-ionic membrane fluidizing compound in a sample for releasing nucleic acid is preferably between about 0.001 and 10% (v/v), more preferably between 0.01% and 5%, most preferably between 0.1 % and 2%. The ultimate concentration of the non-ionic membrane fluidizing compound depends on the nature of the fluidizing compound and the other components of the nucleic acid releasing composition. One skilled in the art can readily determine the proper concentration of membrane fluidizing compound for effective release of nucleic acid from a particular sample by determining binding of a specific probe to nucleic acid released by a particular formulation.
Most non-ionic membrane fluidizing compounds are more soluble in non-aqueous solvents. In such cases, stock solutions can be made in a solvent that is less polar than water, for example, in ethanol or isopropanol.
The aqueous solution of the nucleic acid releasing composition also can include metal chelators such as ethylenediaminetetraacetic acid (EDTA) and ethyleneguaninetetraacetic acid (EGTA). In addition, the aqueous solution can be heated to enhance release of the nucleic acid essentially as described in U.S. Pat. No. 5,837,452 (1988).
The compositions of the present invention are useful for releasing nucleic acid in a non-denatured form suitable for detection of a specific nucleotide sequence. Thus, it is preferred that the nucleic acid releasing compositions be non-denaturing and non-inhibitory of enzymes or proteins used in nucleic acid release, amplification, labeling or detection. This allows the composition to include a labeled or unlabeled nucleic acid probe or primer or other reagents useful in detection of a nucleotide sequence without additional steps to dilute the sample or neutralize denaturing conditions.
In some embodiments, the compositions for releasing nucleic acid also include reagents to label the released nucleic acid for later detection of formed hybrids. Such reagents for labeling nucleic acid comprise a binding ligand comprising a chemical moiety that binds to a nucleic acid and that, when activated by light (i.e., photochemistry), forms at least one covalent bond therewith, a label comprising a detectable moiety and optionally, a binding enhancer comprising a chemical moiety that has a specific affinity for nucleic acids (U.S. patent application Ser. No. 09/265,127).
The photochemical method provides more favorable reaction conditions than the usual chemical coupling method for biochemically sensitive substances. The DNA binding ligand and label can first be coupled and then photoreacted with the nucleic acid, or the nucleic acid can first be photoreacted with the binding ligand and then coupled to the label.
DNA-binding ligands useful herein for linking the nucleic acid component to the label can be any suitable photoreactive form of known DNA-binding ligand. Particularly preferred DNA-binding ligands are intercalator compounds such as the furocoumarins, e.g., angelicin (isopsoralen) or psoralen or derivatives thereof, which photochemically react with nucleic acids, e.g., 4xe2x80x2-aminomethyl-4,5xe2x80x2-dimethylangelicin, 4xe2x80x2-aminomethyl-trioxsalen (4xe2x80x2aminomethyl-4,5xe2x80x2,8-trimethyl-psoralen), 3-carboxy-5- or -8-amino- or -hydroxy-psoralen, as well as mono- or bis-azido aminoalkyl methidium or ethidium compounds.
Particularly useful photoreactive forms of intercalating agents are the azidointercalators. Their reactive nitrenes are readily generated at long wavelength ultraviolet or visible light and the nitrenes of arylazides prefer insertion reactions over their rearrangement products (White, et al., Meth. Enzymol., 46:644 (1977)). Representative intercalating agents include azidoacridine, ethidium monoazide, ethidium diazide, ethidium dimer azide (Mitchell, et al., J. Am. Chem. Soc., 104:4265 (1982)), 4-azido-7-chloroquinoline, and 2-azidofluorene. A specific nucleic acid binding azido compound has been described by Forster, et al., Nucleic Acid Res., 13:745 (1985). Other useful photoreactable intercalators are the furocoumarins which form (2+2) cycloadducts with pyrimidine residues. Alkylating agents also can be used as the DNA binding ligand, including, for example, bischloroethylamines and epoxides or aziridines, e.g., aflatoxins, polycyclic hydrocarbon epoxides, mitomycin and norphillin A.
DNA-binding ligands which do not inhibit nucleic acid amplification enzymes under amplification reaction conditions include, for example, 4xe2x80x2Biotinyl-PEG-4,5xe2x80x2-dimethylangelicin (xe2x80x9cBPAxe2x80x9d), Angelicin-DAPI-Biotin (xe2x80x9cBDAxe2x80x9d), Angelicin-bisbenzimidazole-PEG-azidonitrobenzene (xe2x80x9cAZPIMAxe2x80x9d), Angelicin-bisbenzimidazole-PEG-acridine (xe2x80x9cAPIMAxe2x80x9d), Angelicinbisbenzimidazole-PEG-biotin (xe2x80x9cBPIMAxe2x80x9d) and compounds described in U.S. Pat. Nos. 4,950,744 and 5,026,840. In such compounds, PEG represents any of the known forms of polyethyleneglycol, including pentaoxaheptadecane.
Usually, a stock solution of these compounds is prepared such that an aliquot of the stock solution is added to the reaction mixture to the desired final concentration. The desired concentration can be determined by one skilled in the art using known methods. Such methods include binding studies of the ligand with nucleic acids in a mock clinical sample. The concentration of the labeling reagent in the mixture should be between about 0.001 nanomolar and 10.0 millimolar, preferably between about 0.1 micromolar and 100 micromolar, and most preferably between about 0.1 micromolar and 10 micromolar. The DNA-binding ligand will be present in the aqueous solution of the present invention either as a mixture or as a component of a liposomal formulation.
The label, which is linked to the nucleic acid through the DNA-binding ligand, can be any chemical group or residue having a detectable physical or chemical property, i.e., labeling can be conducted by chemical reaction or physical adsorption. The label includes a functional chemical group to enable it to be chemically linked to the DNA binding ligand. Such labeling materials have been well developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to label DNA as described herein.
Particularly useful labels are enzymatically active groups such as enzymes (Clin. Chem., 22:1243 (1976)), enzyme substrates (British Pat. No. 1,548,741), coenzymes (U.S. Pat. Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (U.S. Pat. No. 4,134,792; fluorescers (Clin. Chem., 25:353 (1979)), and chromophores including phycobiliproteins; luminescers such as chemiluminescers and bioluminescers (Clin. Chem., 25:512 (1979) and ibid, 1531); specifically bindable ligands, i.e., protein binding ligands; antigens; and residues comprising radioisotopes such as 3H, 35S, 32P, 125I, and 14C. Such labels are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
For example, a cofactor-labeled nucleic acid can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. A hapten or ligand (e.g., biotin) labeled nucleic acid can be detected by adding an antibody or an antibody pigment to the hapten or a protein that binds the ligand (e.g., avidin), tagged with a detectable molecule. A detectable molecule has a measurable physical property (e.g, fluorescence or absorbence) or is participant in an enzyme reaction (e.g., see above list). For example, one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property. Examples of the latter include, but are not limited to, betagalactosidase, alkaline phosphatase, papain and peroxidase. For in situ hybridization studies, the final product of the substrate is preferably water insoluble. Other labels, e.g., dyes, will be evident to one having ordinary skill in the art.
If the label is an enzyme, the labeled DNA is ultimately placed in a suitable medium to determine the extent of catalysis. Thus, if the enzyme is a phosphatase, the medium can contain nitrophenyl phosphate and one can monitor the amount of nitrophenol generated by observing the color. If the enzyme is a beta-galactosidase, the medium can contain o-nitro-phenyl-D-galacto-pyranoside, which also liberates nitrophenol. The label can be linked to the DNA binding ligand, e.g., acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines, by direct chemical linkage such as involving covalent bonds, or by indirect linkage such as by the incorporation of the label in a microcapsule or liposome, which in turn is linked to the binding ligand. Methods by which the label is linked to a DNA binding ligand such as an intercalator compound are well known in the art and any convenient method can be used.
Advantageously, the DNA binding ligand is first combined with label chemically and thereafter combined with the nucleic acid component. For example, since biotin carries a carboxyl group, it can be combined with a furocoumarin by way of amide or ester formation without interfering with the photochemical reactivity of the furocoumarin or the biological activity of the biotin. Aminomethylangelicin, psoralen and phenanthridium derivatives can similarly be linked to a label, as can phenanthridium halides and derivatives thereof such as aminopropyl methidium chloride (Hertzberg et al, J. Amer. Chem. Soc., 104:313 (1982)). Alternatively, a bifunctional reagent such as dithiobis succinimidyl propionate or 1,4-butanediol diglycidyl ether can be used directly to couple the DNA binding ligand to the label where the reactants have alkyl amino residues, again in a known manner with regard to solvents, proportions and reaction conditions. Certain bifunctional reagents, possibly glutaraldehyde may not be suitable because, while they couple, they may modify nucleic acid and thus interfere with the assay. Routine precautions can be taken to prevent such difficulties.
The particular sequence used in making the labeled nucleic acid can be varied. Thus, for example, an amino-substituted psoralen can first be photochemically coupled with a nucleic acid, the product having pendant amino groups by which it can be coupled to the label, i.e., labeling is carried out by photochemically reacting a DNA binding ligand with the nucleic acid in the test sample. Alternatively, the psoralen can first be coupled to a label such as an enzyme and then to the nucleic acid.
Advantageously, the DNA binding ligand can be linked to the label by a spacer, which includes a chain of up to about 40 atoms, preferably about 2 to 20 atoms, selected from the group consisting of carbon, oxygen, nitrogen and sulfur. Such spacer can be the polyfunctional radical of a member selected from the group consisting of peptide, hydrocarbon, polyalcohol, polyether, polyamine, polyimine and carbohydrate, e.g., -glycyl-glycyl-glycyl- or other oligopeptide, carbonyl dipeptides, and omega-amino-alkane-carbonyl radical or the like. Sugar, polyethylene oxide radicals, glyceryl, pentaerythritol, and like radicals also can serve as spacers. Spacers can be directly linked to the nucleic acid-binding ligand and/or the label, or the linkages may include a divalent radical of a coupler such as dithiobis succinimidyl propionate, 1,4-butanediol diglycidyl ether, a diisocyanate, carbodiimide, glyoxal, glutaraldehyde, or the like.
Nucleic acid labeling reagents including the binding ligand and label also optionally can include a binding enhancer as described U.S. application Ser. No. 09/265,127. Covalent or non-covalent complexes of a binding ligand, a binding enhancer and a label is referred to herein as a xe2x80x9cLAC.xe2x80x9d
The nucleic acid binding enhancer (xe2x80x9cbinding enhancerxe2x80x9d), serves to enhance the affinity of the LAC for nucleic acids above that exhibited with the binding ligand alone. Accordingly, binding enhancers tend to have a specific affinity for nucleic acids when compared to non-nucleic acid sample/reaction constituents. The binding enhancer can be the same as or different from the binding ligand. In other words, the binding ligand and the binding enhancer can each be an intercalator, wherein one of the two is a monoadduct-forming species, and the other is present to enhance binding by this monoadduct-forming species. Examples of such xe2x80x9cdual rolexe2x80x9d binding ligands are described in Chaires, et al., J. Med. Chem., 40:261-266 (1977). Therein, it has been described that binding of a bis-intercalating anthracycline antibiotic reached as high as 1011 at 20xc2x0 C. It was also shown that the affinity of a similar monointercalator is not above 107 (Chaires, et al., Biochem., 35:2047-2053 (1996)).
The binding enhancer also can be a non-intercalating compound. There are many non-intercalating nucleic acid binding molecules known in the art. A bis-benzimidazole derivative commonly known as Hoechst 33258 has shown affinity as high as 3.2xc3x97108Mxe2x88x921(Haq, et al., J. Mol. Biol., 271:244-257 (1997)). Other non-intercalating binding enhancers are oligo pyrroles, phenyl indole derivatives and the like. These molecules do not bind nucleic acids solely on the basis of positive charge. Other suitable binding enhancers bind nucleic acids on the basis of hydrogen bond formation, hydrophobic interaction in the major or minor groove of the nucleic acid double helix and other non-ionic interactions that give rise to high affinity reactions with nucleic acids.
Not every compound capable of forming an electrostatic bond with a negatively charged nucleic acid can serve as a binding enhancer. For example, polycations such as polyamines are generally not suitable for use in the present invention because of their inability to specifically bind to nucleic acids in crude samples and in the presence of amplification reaction components. Such positively charged compounds can, for example, non-specifically bind to all anionic macromolecules present in the sample, and not just to nucleic acids. In addition, the binding enhancer should be capable of specifically binding to nucleic acids in the presence of 10 to 20 mM magnesium, which is typically required for most amplification reactions. At this concentration, compounds that bind to nucleic acids solely on the basis of electrostatic interactions do not form stable complexes with nucleic acids and thus require a greater concentration of LAC for efficient labeling.
As discussed above, the binding ligand for labeling nucleic acid is either directly or indirectly linked to a label. Such attachment can be either covalent or ionic, so long as it is stable under the conditions in which the LAC is employed. Chemical attachments can be accomplished by any of a variety of well known methods. For example, if the binding ligand contains or is derivatised to contain an available carboxyl group and the label contains or is derivatized to contain an available amino group, the two can be reacted together to form an ester linkage. By xe2x80x9cavailablexe2x80x9d, it is meant that the formation of a linkage will not interfere with the functioning of the label (i.e., its ability to be detected or to catalyze a detectable reaction) or the ligand (i.e., it""s ability to bind nucleic acids). Particularly useful labels are enzymes, enzyme substrates, fluorophore, radioisotopic compounds, chromophores, magnetically responsive compounds, antibody epitope-containing compounds, haptens, and the like.
The binding ligand, binding enhancer, and label or labeling nucleic acid can also be indirectly attached via a linker. Such linkers are specifically designed to promote efficient binding of the binding ligand to the nucleic acids and functioning of the label attached thereto. This occurs by providing adequate physical separation between the two components of the LAC to prevent interference of one by the other. The use of linkers is described generally in U.S. Pat. Nos. 4,582,789 and 5,026,840. Certain compounds can serve the dual role of a binding enhancer and a linker. For example, linkers can be constructed from positively charged compounds, such that they enhance binding with negatively charged nucleic acids. However, in order for the linker to also serve as a binding enhancer, it is necessary for it to have a specific affinity for nucleic acids, and not just a structure specific electrostatic affinity for negatively charged compounds. The polyalkylarnine linkers described in U.S. Pat. No. 5,026,840 are not optimal as binding enhancers but are suitable as linkers.
In a preferred embodiment, a bifunctional linker is used that is capable of reacting with both the nucleic acid binding moiety and the label to form a chemical bridge therebetween. However, in an alternate embodiment, a multifunctional linker can be employed, to which the binding ligand, the binding enhancer and the label are attached as a xe2x80x9cbranchedxe2x80x9d complex. Such complex formats and chemical reactions for forming these types of complexes are well known in the art.
Compositions comprising an aqueous solution for releasing nucleic acid of the present invention having the appropriate combination of nucleic acid releasing, labeling and detecting reagents to achieve single step processing and detection also are provided herein. Such compositions require that all the components of the composition not be denaturing or inhibitory to enzymes or proteins used in nucleic acid release, amplification, labeling or detection. All these components when mixed to produce the final reagent are delivered to the sample in an aqueous solution which can be water or a buffer solution pH of which is preferably between 3 and 12. More preferably between 5 and 10 such that the released nucleic acids are not substantially degraded. The particular reagents to be added and their optimal concentration depends on various factors including the nature of the sample and the particular reagents chosen. One skilled in the art can readily select the proper reagents and determine an optimal concentration of each without resort to undue experimentation.
The present invention also provides methods and kits for using the disclosed compositions in assays for detecting the presence of a nucleotide sequence in nucleic acid of a sample containing cells. Such assays are used for diagnosis of infectious diseases, cancer, human genetic disorders, and others like histocompatibility (e.g., HLA) typing, forensic and paternity testing. For example, by contacting and treating the sample with the above described compositions that contain reagents for releasing nucleic acid from cells and appropriate labeling reagents (e.g., LACs), the samples can be used for hybridization diagnosis without any further processing of the sample. Thus, a urine sample, for instance, that is suspected of bacterial infections can be labeled without centrifugation, filtration or dialysis and the cells in the samples are lysed without any separation step.
Test samples include body fluids, e.g., urine, blood, semen, cerebrospinal fluid, pus, amniotic fluid, tears, or semisolid or fluid discharge, e.g., sputum, saliva, lung aspirate, vaginal or urethral discharge, stool or solid tissue samples, such as a biopsy or chorionic villi specimens. Test samples also include samples collected with swabs from the skin, genitalia, or throat. The compositions of the invention can be added directly to the sample or to cells isolated from the sample.
The assay method can detect the nucleic acid from essentially any species of organism, including, for example, Acintobacter, Actinomyces, Aerococcus, Aeromonas, Alclaigenes, Bacillus, Bacteriodes, Bordetella, Branhamella, Bevibacterium, Campylobacter, Candida, Capnocytophagia, Chlamydia, Chromobacterium, Clostridium, Corynebacterium, Cryptococcus, Deinococcus, Enterococcus, Erysielothrix, Escherichia, Flavobacterium, Gemella, Gonorrhea, Haemophilus, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leuconostoc, Listeria, Micrococcus, Mycobacterium, Neisseria, Nocardia, Oerskovia, Paracoccus, Pediococcus, Peptostreptococcus, Propionibacterium, Proteus, Psuedomonas, Rahnella, Rhodococcus, Rhodospirillium, Staphlococcus, Streptomyces, Streptococcus, Vibrio, and Yersinia. Also included are viruses such as the hepatitis viruses and human immunodeficiency viruses (HIV).
The present methods also can be used to detect nucleic acid from eukaroytes (protists) in samples from higher organisrns, such as animals or humans. Eukaroytes include algae, protozoa, fungi and slime molds. The term xe2x80x9calgaexe2x80x9d refers in general to chlorophyll-containing protists, descriptions of which are found in Smith, Cryptogamic Botany, 2nd ed. Vol. 1, Algae and Fungi, McGraw-Hill, (1955). Eukaryotic sequences according to the present invention includes all disease sequences. Accordingly, the detection of genetic diseases, for example, also are embraced by the present invention.
Methods of detecting a nucleotide sequence involve contacting the above described aqueous compositions for releasing nucleic acid with a sample suspected of containing the nucleotide sequence of interest. The mixture is incubated for an appropriate period of time and under conditions suitable for releasing the nucleic acid from the cells. If release and detection of the nucleic acid is sought as a single step, the nucleic acid releasing composition also includes one or more nucleic acid probes or primers that are complementary to the nucleotide sequence to be detected and other reagents depending on the detection format to be used. Such nucleic acid primers or probes can be an oligonucleotide or, in some cases, a larger nucleic acid molecule.
If the sample already contains released or isolated nucleic acid, the incubation period can be between a few seconds to five min. When the sample contains whole cells, incubation between two minutes (min) to two hours (xe2x80x9chrsxe2x80x9d) may be necessary.
Amplification methods suitable for use with the present methods include, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription mediated amplification (TMA) reaction, nucleic acid sequence based amplification (NASBA) reaction, and strand displacement amplification (SDA) reaction. These methods of amplification are well known in the art.
PCR can be performed as according to Whelan, et al, J. Clin. Microbiol., 33(3):556-561 *(1995). Briefly, a PCR reaction mixture includes two specific primers, dNTP, 0.25 Units (U) of Taq polymerase, and 1xc3x97PCR Buffer. For every 25 xcexcl PCR reaction, a 2 xcexcl sample (e.g., isolated DNA from target organism) is added and amplified on a thermal cycler. The amplification cycle includes an initial denaturation, and up to 50 cycles of annealing, strand elongation and strand separation (denaturation).
LCR can be performed as according to Moore, et al., J. Clin. Microbiol., 36(4):1028-1031 (1998). Briefly, an LCR reaction mixture contains two pair of probes, dNTP, DNA ligase and DNA polymerase representing about 90 xcexcl, to which is added 100 xcexcl of isolated nucleic acid from the target organism. Amplification is performed in a thermal cycler (e.g., LCx(copyright) thermal cycler, Abbott Labs, North Chicago, Ill.).
SDA can be performed as according to Walker, et al., Nucleic Acids Res., 20(7):1691-1696 (1992). Briefly, an SDA reaction mixture contains four SDA primers, dGTP, dCTP, TTP, dATPS, 150 U of Hinc II, and 5 U of exonucrease deficient E. coli DNA polymerase I. The sample mixture is heated 95xc2x0 C. for 4 min to denature target DNA prior to addition of the enzymes. After addition of the two enzymes, amplification is carried out for 120 min. at 37xc2x0 C. in a total volume of 50 xcexcl. The reaction is terminated by heating for 2 min at 95xc2x0 C.
NASBA can be performed as according to Heim, et al., Nucleic Acids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction mixture contains two specific primers, dNTP, NTP, 6.4 U of AMV reverse transcriptase, 0.08 U of Escherichia coli Rnase H, and 32 U of T7 RNA polymerase. The amplification is carried out for 120 min at 41xc2x0 C. in a total volume of 20 xcexcl.
TMA can be performed as according to Wylie, et al., Journal of Clinical Microbiology, 36(12):3488-3491 (1998). In TMA, nucleic acid targets are captured with magnetic beads containing specific capture primers. The beads with captured targets are washed and pelleted before adding amplification reagents, which contain amplification primers, dNTP, NTP, 2500 U of reverse transcriptase and 2500 U of T7 RNA polymerase. A 100 xcexcl TMA reaction mixture is placed in a tube, 200 xcexcl oil reagent is added and amplification is accomplished by incubation at 42xc2x0 C. in a waterbath for one hour (xe2x80x9chrxe2x80x9d).
A variety of amplification enzymes are well known in the art and include, for example, DNA polymerase, RNA polymerase, reverse transcriptase, Q-beta replicase, thermostable DNA and RNA polymerases. Because these and other amplification reactions are catalyzed by enzymes, it is important for a single step assay that the nucleic acid releasing reagents and the detection reagents are not potential inhibitors of amplification enzymes if the ultimate detection is to be amplification based.
Also included in the composition for amplification are appropriate nucleoside triphosphates, amplification buffer and certain ions. The concentrations of nucleic acid primers and enzymes can be selected for specific use. For example, for polymerase chain reaction, the concentration of the nucleic acid primer is between 1 picomole and 1 millimole when added to the sample. The enzyme concentration can vary between about 0.01 U and 100,000U. One skilled in the art can determine the optimal concentration of enzyme and other reagents by routine experimentation.
Detection of the nucleotide sequences also can be performed directly without amplification by hybridizing the sample nucleic acid to the nucleic acid probe present in the composition. In this case, the nucleic acid is contacted and incubated with the labeling reagents (provided in the nucleic acid release composition or separately) and the mixture is irradiated at a particular wavelength for the covalent interaction between the photochemically reactive DNA binding ligand and the test sample to take place. After labeling, the material is hybridized under specified hybridization conditions with a probe specific for the target nucleic acid.
Hybridization of the labeled sample nucleic acid or the labeled nucleic acid probe can be detected in any conventional hybridization assay format and, in general, in any format suitable for detecting the hybridized product or aggregate comprising the labeled nucleic acid. If the sample nucleic acid has been labeled, it can be used for hybridization in solution and solid-phase formats, including, in the latter case, formats involving immobilization of either sample or nucleic acid probe. For example, preimmobilized nucleic acid probe can be hybridized with labeled sample nucleic acid. The presence of label associated with the solid phase indicates hybridization between the probe and the sample nucleic acid and, thus, detection of the target nucleotide sequence. Alternatively, unlabeled sample nucleic acid can be preimmobilized and a labeled probe evaluated for hybridization thereto.
Preferable concentration for the probe is between about 0.01 picomole and 10 millimoles, more preferably between about 1 picomole and 1 millimole, and most preferably between about 10 picomole and 10 micromoles. Methods of detecting hybrids on solid phases are well known in the art and have been extensively described (e.g., U.S. Pat. Nos. 5,232,831, 4,950,613, 486,539 and 4,563,419).
The nucleic acid probe comprises at least one hybridizable, e.g., singlestranded, base sequence substantially complementary to or homologous with the nucleotide sequence to be detected. However, such base sequence need not be a single continuous polynucleotide segment, but can comprise two or more individual segments interrupted by non-homologous sequences. These non-homologous sequences can be linear or they can be self-complementary and form hairpin loops. In addition, the homologous region of the probe can be flanked at the 3xe2x80x2- and 5xe2x80x2 termini by non-homologous sequences, such as those comprising the DNA or RNA or a vector into which the homologous sequence had been inserted for propagation. In either instance, the probe as presented as an analytical reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular hybridizable, e.g., single-stranded polynucleotides can be used as the probe element, with major or minor portions being duplexed with a complementary polynucleotide strand or strands, provided that the critical homologous segment or segments are in single-stranded form and available for hybridization with sample DNA or RNA. Useful probes include linear or circular probes wherein the homologous probe sequence essentially is a single-stranded form (Hu et al., Gene, 17:271 (1982)).
The nucleic acid probe can be used in any conventional hybridization technique. As improvements are made and conceptually new formats are developed, such can be readily applied to the present probes. Conventional hybridization formats that are particularly useful include those wherein the sample nucleic acids or the polynucleotide probe are immobilized on a solid support (solid-phase hybridization) and those wherein the polynucleotide species are all in solution (solution hybridization).
In solid-phase hybridization formats, one of the polynucleotide species participating in hybridization is fixed in an appropriate manner in its singlestranded form to a solid support. Useful solid supports are well known in the art and include those, for example, which bind nucleic acids either covalently or non-covalently. Non-covalent binding supports, which are generally understood to involve hydrophobic bonding include naturally occurring and synthetic polymeric materials, such as nitrocellulose, derivatized nylon and fluorinated polyhydrocarbons, in a variety of forms such as filters, beads or solid sheets. Covalent binding supports (in the form of filters, beads or solid sheets, just to mention a few) are also useful and comprise materials having chemically reactive groups or groups such as dichlorotriazine, diazobenzyloxymethyl, and the like, which can be activated for binding to polynucleotides.
It well known that non-covalent immobilization of an oligonucleotide to a solid support such as nitrocellulose paper is generally ineffective for detecting hybridization. Thus, covalent immobilization is preferred and can be achieved by phosphorylation of an oligonucleotide by a polynucleotide kinase or by ligation of the 5xe2x80x2-phosphorylated oligonucleotide to produce multi-oligonucleotide molecules capable of immobilization. The conditions for kinase and ligation reaction have been described previously (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1.53 and 5.33 (1989). Thus oligonucleotide probes specific for genetic defects related to hemoglobinopathies, such as sickle cell anemia and alpha-thalassemias can be immobilized on nitrocellulose paper and contacted with patient sample nucleic acid labeled by the above described method. The photochemical labeling can be done in a single step without the need to obtain purified nucleic acid samples and without affecting the specific hybridizability of the labeled sample.
A typical solid-phase hybridization technique begins with immobilization of sample nucleic acids onto the support in single-stranded form. This initial step essentially prevents reannealing of complementary strands from the sample and can be used for concentrating sample material on the support for enhanced delectability. The nucleic acid probe is then contacted with the support and hybridization detected by measurement of the label as described herein. The solid support provides a convenient means for separating labeled probe, which has hybridized to the sequence to be detected, from probe that has not hybridized.
Another method of interest is the sandwich hybridization technique wherein one of two mutually exclusive fragments of the homologous sequence of the probe is immobilized and the other is labeled. The presence of the polynucleotide sequence of interest results in dual hybridization to the immobilized and labeled probe segments (G. Rankim, et al., 21:77-85 (1983)).
In one embodiment, the immobile phase of the hybridization system can be a series or matrix of spots of known kinds and/or dilutions of denatured DNA. This can be prepared by pipetting appropriate small volumes of native DNA onto a dry nitrocellulose or nylon sheet, floating the sheet on a sodium hydroxide solution to denature the DNA, rinsing the sheet in a neutralizing solution, then baking the sheet to fix the DNA. Before DNA:DNA hybridization, the sheet is usually treated with a solution that inhibits non-specific binding of added DNA during hybridization.
In solid phase detection systems, unhybridized labeled test sample can be removed by washing following hybridization. After washing, the hybrid is detected through the label carried by the test sample, which is specifically hybridized with a specific probe.
The present invention further features kits that incorporates the components of the invention and makes possible convenient performance of the invention. Such kit may also include other materials that would make the invention a part of other procedures including adaptation to multi-well technologies. The items comprising the kit may be supplied in separate vials or may be mixed together, where appropriate.